Formation of cell sheet on acellular porcine corneal stroma with human corneal endothelial cells cocultured by mouse embryonic stem cell conditioned medium

Abstract

Background Corneal transplantation faces a great challenge because of the shortage of corneal donors and difficulty of human corneal endothelial cells (HCECs) regeneration in vitro.So the study on tissue engineering cornea is still a main topic.Previous research showed that mouse embryonic stem cell conditioned medium (ESC-CM) improved the proliferative capacity of HCECs in vitro, and acellular porcine corneal stroma (APCS) was a good saffold material.However, whether HECEs cultured by mouse ESC-CM can form cell sheet in vitro were rarely studied. Objective This study was to investigate the potential that HCECs cultured by mouse ESC-CM form a monolayer cell sheet. Methods The supernatant of ESC-CM was collected after mouse ES-E14 cells were cultured, and the cultured medium was centrifuged and mixed with 75% human corneal endothelium medium (CEM) at a proportion of 1∶3 to prepare the 25% ESC-CM system.Primary cultures of HCECs were established from explants of corneal limbal with Descemet's membrane, and the cells were identified by using reverse-transcription PCR to determine the expressions of collagen Ⅷ (Col Ⅷ) mRNA and neuron-specific enolase (NSE) mRNA in the cells.APCS was prepared by decellularization with phospholipase A2 and bicarbonate solution, and the second generation of HCECs were inoculated on the sterilized APCS at a 800/mm2 density.The morphology of the cells was observed by hematoxylin-eosin staining under the phase-contrast microscope.The expressions of zona occludens protein-1 (ZO-1) and Na+ -K+ -ATPase in the cell sheet were detected by immunofluorescence staining. Results The second generation of HCECs cultured with 25% ESC-CM in vitro showed the hexagon in shape with positive expressions for Col Ⅷ mRNA and NSE mRNA.Decellularization APCS was transparent, and no corneal cells were seen, the structures of corneal collagenous fibres were regular.HCECs attached closely to APCS and formed monolayer sheet 7 days after culture on the APCS with the cell density of (2 694±143)/mm2.ZO-1 and Na+ -K+ -ATPase were positively expressed on the HCECs sheet. Conclusions Twenty-five percent ESC-CM promotes the proliferation and maintains the normal morphology of HCECs.APCS provide a good scoffold and microenvironment for the formation of HCEC sheet.The HCEC sheet Possesses the pump function of HCECs and is a good corneal donor for transplantation. Key words: Corneal stroma/transplantation; Endothelial cells, corneal/cytology; Humans; Tissue engineering/methods; Tissue scaffolds; Culture media, conditioned; Embryonic stem cells, mouse; Acellular corneal stroma, porcine